The use of dry chemistry techniques in the measurement of specific analytes in biological fluids such as urine and blood has attained prime importance. Among other advantages dry chemistry, or solid phase methods, permit rapid analysis by relatively unskilled personnel, require no reagent preparation, and provide economy of both reagents and sample thus making them ideally suited for patient self-testing, among other uses. In solid phase chemistry, reagents are present in dry form, most frequently in an impregnated matrix. Further, the reagents are normally unitized, i.e., the reagents for each individual test are used and disposed of as a single entity. One convenient and popular form is that of a test strip in which the reagent matrix is affixed to a carrier that supports the reaction area and can also serve as a handle. When the liquid sample, typically blood or urine, which contains the analyte of interest is brought into contact with the test area, the reagents located therein are immediately activated and produce a reaction or sequence of reactions, resulting usually in the formation of a color which can be compared by visual means to a standard color chart or measured more precisely by an instrument such as a reflectance photometer.
One test which is exemplary of the type of assay performable with dry chemistry test devices is the determination of blood glucose, which is important, inter alia, in monitoring diabetic and hypoglycemic patients. A typical test strip, for example, contains the enzymes glucose oxidase and a peroxidase, as well as an indicator such as 3,3',5,5'-tetramethylbenzidine, also referred to as "TMB". If glucose is present in the sample, it reacts with oxygen in the presence of glucose oxidase to form gluconic acid and hydrogen peroxide. The peroxide, in turn, reacts with the TMB in the presence of the peroxidase, oxidizing the TMB. In reduced form, TMB, is colorless; however, when it is oxidized, TMB is blue, red or purple, depending upon the charge transfer complex formed. Thus, one can determine if glucose is present, and in what quantity, by observing the formation of color. This reaction system is analyte specific, because hydrogen peroxide is not a normal component of the sample, and it only forms when the glucose-specific enzyme, glucose oxidase, acts on its substrate.
In its simplest form, a test device of the type used in dry chemistry analyses of the type monitored by diffuse reflectance methods comprises three functional zones or layers. The first, which plays no actual part in the assay, is a support or carrier layer. This layer is inert, impervious to liquids, and is generally constructed from rigid materials such as thermoplastic films. It may be transparent or opaque, depending upon the side through which the reaction is to be observed. A second, reflective layer, which can be a distinctly defined layer or may be incorporated with the carrier material, the reagent layer, or both, may be employed to aid in reading the detectable signal when it forms. This is especially helpful in systems which use a reflected light detector. The reflective layer contains some material which reflects light not absorbed by the detectable signal generator and generally takes the form of a coating, foam, membrane, paper, or metal foil which is reflective. Substances useful for this purpose include pigments such as titanium dioxide, zirconium dioxide, zinc oxide and barium sulfate.
The third functional layer, which can be generally referred to as the reagent layer, contains the reactive ingredients which cause formation of the detectable signal. It is this layer which is actually observed or measured to determine the analyte. Various enzymes, substrates, receptors, and binding partners can be incorporated in this layer, such as labeled antibodies, indicator molecules, fluorescent agents, capping or quenching agents, and so forth.
The sample for analysis is commonly applied by dropping from above the reagent layer, whereupon it makes contact with a spreading or metering layer which is positioned over the reagent layer. In the case of a whole blood sample, the spreading layer will serve also to separate the highly colored red cells from the plasma, which would otherwise interfere in the reaction. The plasma soaks or diffuses into the reagent matrix where the reactions occur. In most types of these soak-through devices, the residual blood sample and cells must be wiped or rinsed from the surface of the test area in order to measure color formation. Alternatively, color may be measured from the bottom of the reaction area provided that, of course, the support upon which the reation matrix rests is formed from a clear, transparent material. A second type of analytical device functions by a relatively horizontal, or parallel, rather than vertical sample flow whereby the sample moves across the top of the reaction area, such as by capillary action, gravity or other forces. As the sample travels over the surface of the reaction area carrying red blood cells and other cellular elements with it, the plasma moves into the reagent layer itself, where it reacts to form a detectable moiety.
Two distinct approaches have been used to construct the zone or layer containing the reagents for the assay. The first approach involves the use of a paper, e.g., cellulose, or synthetic fiber matrix which has been saturated with solutions containing the assay reagents and then dried. In the second approach, the reagent layer is constructed by casting a porous film containing all the required assay components. In the case of constituents which require segregation before use, the reagents may be deposited in several film layers, so that the reagent layer created thereby is actually constructed of multiple film layers, each of which contains a portion of the assay components required. Typically, membranes which separate or trap cellular components from the blood are also used. The quantities of reagents made available during analysis are controlled by their concentration in the casting medium, the thickness of the cast film and their solubility in the sample solvent.
A primary advantage of these films is the high degree of uniformity of the reagent layer. With the use of reagent films and of reflectance photometry, it is now possible to achieve measurement results with dry chemistry strips of the same precision and accuracy as with wet chemistry methods. In the case of these test strips, there is involved mixing a solution or suspension of the reagents and applying the mix to a test strip, followed by evaporation of the liquid. An aqueous solution, or a solution containing a dispersed polymeric binder, is typically employed. These aqueous solution-based systems are problematic for several important reasons. First, many important substances, including TMB and other indicators, are insoluble in water. This, taken with other problems of hydrolytic stability of the reagents and compatibility of the reagents, renders many substances unsuitable for use in test strip devices. Also, if enzymes and substrates, for instance, are mixed in a solution, they will react prior to application of the analyte, with resulting failure of the system. As was pointed out, the liquid phase, such as water, has to be removed from these systems, and this must be done at elevated temperatures to remove all the moisture. Generally, the parameters used for evaporation are 55.degree. C. or higher for a minimum of 20 to 30 minutes, which conditions are sufficient to cause some damage to most enzymatic systems. Thus there is a pressing need to develop techniques in which water is eliminated.